Antibiotic-resistant Gram-positive bacteria, such as Enterococcus faecalis and Staphylococcus aureus, are major causes of hospital-acquired infections. E. faecalis is a successful hospital-acquired pathogen partly due to its intrinsic resistance to commonly used antibiotics that target bacterial cell envelope biogenesis. However, many questions regarding the genetic and biochemical basis for intrinsic antimicrobial resistance in E. faecalis remain unanswered. Preliminary studies identified a new signal transduction system containing a eukaryotic-type Ser/Thr kinase (PrkC) that is required for intrinsic antimicrobial resistance in E. faecalis. We hypothesize that PrkC monitors the cell envelope for perturbations caused by envelope-active antibiotics and mediates an adaptive biological response to produce antimicrobial resistance. Our long-term goal is to understand the role of this new signaling system in mediating resistance, and more generally, the genetic and biochemical basis for intrinsic antimicrobial resistance in E. faecalis. The objective of this proposal is to begin to elucidate the role of signal transduction, and specifically of the PrkC pathway, in mediating intrinsic antimicrobial resistance in enterococci. To achieve this goal, we will: 1) Define the PrkC-dependent regulatory circuit; 2) Identify direct substrates for phosphorylation by PrkC; and 3) Characterize effectors of intrinsic antimicrobial resistance (the output of PrkC-dependent signaling). Thus, the research proposed here will provide insights into the function of a new bacterial signaling system, enhance fundamental understanding of the mechanisms responsible for antimicrobial resistance in Gram- positive bacteria, and form the foundation for future efforts to develop innovative therapies against infections caused by resistant bacteria.